The effect of off-bottom versus on-bottom oyster culture on total and pathogenic Vibrio spp. abundances in oyster tissue, water and sediment samples.

Varying culture methods are commonly used for eastern oyster, Crassostrea virginica, aquaculture in the Northeast United States. Vibrio vulnificus and V. parahaemolyticus, two human pathogenic bacteria species, accumulate in this edible, filter feeding shellfish. This study examined the use of two methods in an intertidal area (oysters cultured in trays and in bags on sediment) and two methods in a subtidal area (oysters cultured in trays and loose on the sediment) in Massachusetts over the growing season in 2015. Abundance of total V. vulnificus along with total and pathogenic (tdh+/trh+) V. parahaemolyticus were determined in oysters, sediment and water using real-time PCR. Temperature, salinity, turbidity and chlorophyll were continually measured every 15 min at each location. There were significantly higher abundances of total and pathogenic V. parahaemolyticus in on-bottom cultured oysters, while significantly higher abundances of V. vulnificus were identified in oysters from off-bottom culture in a subtidal location in Duxbury Bay, MA. In an intertidal location, Wellfleet Bay, MA, significantly higher abundances of total and tdh+V. parahaemolyticus were found in off-bottom oysters, but significantly higher abundances of V. vulnificus and trh+V. parahaemolyticus were found in on-bottom oysters. Spearman's correlation indicated that temperature is positively associated with concentrations of Vibrio spp. in oysters, water and sediment, but positive correlations between salinity and Vibrio spp. was also observed. Conversely, turbidity had a negative effect on Vibrio spp. concentrations in all sample types. There was no observed relationship inferred between chlorophyll and Vibrio spp. abundances in oysters, water or sediment.

Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea virginica.

Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits.IMPORTANCE Despite their biological and ecological significance, a mechanistic characterization of microbiome function is frequently missing from many nonmodel marine invertebrates. As an initial step toward filling this gap for the eastern oyster, Crassostrea virginica, this study provides an integrated taxonomic and functional analysis of the oyster microbiome using samples from a coastal salt pond in August 2017. The study identified high variability of the microbiome across tissue types and among individual oysters, with some dominant taxa showing higher relative abundance in specific tissues. A high prevalence of Mollicutes in the adult oyster gut was revealed by comparative analysis of the gut, biodeposit, and larva microbiomes. Phylogenomic analysis and metabolic reconstruction suggested the oyster-associated Mollicutes is closely related but functionally distinct from Mollicutes isolated from other marine invertebrates. To the best of our knowledge, this study represents the first metagenomics-derived functional inference of Mollicutes in the eastern oyster microbiome.

Embryonic exposures to mono-2-ethylhexyl phthalate induce larval steatosis in zebrafish independent of Nrf2a signaling.

Mono-2-ethylhexyl phthalate (MEHP) is the primary metabolite of the ubiquitous plasticizer and toxicant, di-2-ethylhexyl phthalate. MEHP exposure has been linked to abnormal development, increased oxidative stress, and metabolic syndrome in vertebrates. Nuclear factor, Erythroid 2 Like 2 (Nrf2), is a transcription factor that regulates gene expression in response to oxidative stress. We investigated the role of Nrf2a in larval steatosis following embryonic exposure to MEHP. Wild-type and nrf2a mutant (m) zebrafish embryos were exposed to 0 or 200 µg/l MEHP from 6 to either 96 (histology) or 120 hours post fertilization (hpf). At 120 hpf, exposures were ceased and fish were maintained in clean conditions until 15 days post fertilization (dpf). At 15 dpf, fish lengths and lipid content were examined, and the expression of genes involved in the antioxidant response and lipid processing was quantified. At 96 hpf, a subset of animals treated with MEHP had vacuolization in the liver. At 15 dpf, deficient Nrf2a signaling attenuated fish length by 7.7%. MEHP exposure increased hepatic steatosis and increased expression of peroxisome proliferator-activated receptor alpha target fabp1a1. Cumulatively, these data indicate that developmental exposure alone to MEHP may increase risk for hepatic steatosis and that Nrf2a does not play a major role in this phenotype.

Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the zebrafish, Danio rerio.

Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.

Lesion bacterial communities in American lobsters with diet-induced shell disease.

In southern New England, USA, shell disease affects the profitability of the American lobster Homarus americanus fishery. In laboratory trials using juvenile lobsters, exclusive feeding of herring Clupea harengus induces shell disease typified initially by small melanized spots that progress into distinct lesions. Amongst a cohabitated, but segregated, cohort of 11 juvenile lobsters fed exclusively herring, bacterial communities colonizing spots and lesions were investigated by denaturing gradient gel electrophoresis of 16S rDNA amplified using 1 group-specific and 2 universal primer sets. The Bacteroidetes and Proteobacteria predominated in both spots and lesions and included members of the orders Flavobacteriales (Bacteriodetes), Rhodobacterales, Rhodospirillales and Rhizobiales (Alphaproteobacteria), Xanthomonadales (Gammaproteobacteria) and unclassified Gammaproteobacteria. Bacterial communities in spot lesions displayed more diversity than communities with larger (older) lesions, indicating that the lesion communities stabilize over time. At least 8 bacterial types persisted as lesions developed from spots. Aquimarina 'homaria', a species commonly cultured from lesions present on wild lobsters with epizootic shell disease, was found ubiquitously in spots and lesions, as was the 'Candidatus Kopriimonas aquarianus', implicating putative roles of these species in diet-induced shell disease of captive lobsters.

Combined effects of a parasite, QPX, and the harmful-alga, Prorocentrum minimum on northern quahogs, Mercenaria mercenaria.

Northern quahogs, Mercenaria mercenaria (L.), frequently are infected with the parasite Quahog Parasite Unknown (QPX, Labyrintohomorpha, Thraustochytriales), which can cause morbidity and mortality of the quahogs. Possible interactions between this parasitic disease and exposure to the harmful dinoflagellate Prorocentrum minimum in M. mercenaria were studied experimentally. Quahogs from Massachusetts with variable intensity of QPX infection were exposed, under controlled laboratory conditions, to cultured P. minimum added to the natural plankton at a cell density equivalent to a natural bloom. After 5 days of exposure, individual clams were diagnosed histologically to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Further, cellular defense status of clams was evaluated by analyzing hemocyte parameters (morphological and functional) using flow-cytometry. Exposure of quahogs to P. minimum resulted in: a lower percentage of phagocytic hemocytes, higher production of reactive oxygen species (ROS), larger hemocyte size, more-numerous hemocytic aggregates, and increased numbers of hemocytes in gills accompanied by vacuolation and hyperplasia of the water-tubular epithelial cells of the gills. Quahogs had a low prevalence of QPX; by chance, the parasite was present only in quahogs exposed to P. minimum. Thus, the effect of QPX alone on the hemocyte parameters of quahogs could not be assessed in this experiment, but it was possible to assess different responses of infected versus non-infected quahogs to P. minimum. QPX-infected quahogs exposed to P. minimum had repressed percentage of phagocytic hemocytes, consistent with immuno-modulating effect of P. minimum upon several molluscan species, as well as smaller hemocytes and increased hemocyte infiltration throughout the soft tissues. This experiment demonstrates the importance of considering interactive effects of different factors on the immunology and histopathology of bivalve shellfish, and highlights the importance of considering the presence of parasites when bivalves are subjected to harmful-algal blooms.

Finfish and aquatic invertebrate pathology resources for now and the future.

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology.

Host susceptibility hypothesis for shell disease in American lobsters.

Epizootic shell disease (ESD) in American lobsters Homarus americanus is the bacterial degradation of the carapace resulting in extensive irregular, deep erosions. The disease is having a major impact on the health and mortality of some American lobster populations, and its effects are being transferred to the economics of the fishery. While the onset and progression of ESD in American lobsters is undoubtedly multifactorial, there is little understanding of the direct causality of this disease. The host susceptibility hypothesis developed here states that although numerous environmental and pathological factors may vary around a lobster, it is eventually the lobster's internal state that is permissive to or shields it from the final onset of the diseased state. To support the host susceptibility hypothesis, we conceptualized a model of shell disease onset and severity to allow further research on shell disease to progress from a structured model. The model states that shell disease onset will occur when the net cuticle degradation (bacterial degradation, decrease of host immune response to bacteria, natural wear, and resorption) is greater than the net deposition (growth, maintenance, and inflammatory response) of the shell. Furthermore, lesion severity depends on the extent to which cuticle degradation exceeds deposition. This model is consistent with natural observations of shell disease in American lobster.

Induction of cetacean cytochrome P4501A1 by beta-naphthoflavone exposure of skin biopsy slices.

Marine mammals can accumulate environmental contaminants in their blubber at concentrations harmful to laboratory animals. Induction of the cytochrome P450 1A1 (CYP1A1) enzyme is widely used as a biomarker of exposure and molecular effects in animal species, yet the validity of this biomarker has not been established in marine mammals. In vivo studies are generally precluded in these protected species, but skin biopsies (epidermis and dermis) can be collected in a minimally invasive way. We developed an in vitro assay using skin biopsy slices to examine CYP1A1 protein induction in marine mammals in response to chemical exposure. Skin biopsies from sperm whale (Physeter macrocephalus) were exposed for 24 h to beta-naphthoflavone (BNF), a prototypical CYP1A1 inducer, and CYP1A1 induction was detected by immunohistochemical staining in endothelial cells, smooth muscle cells, and fibroblasts. Biopsy slices were exposed to a range of BNF concentrations (0.6-600 microM), and a significant concentration-effect relationship was observed in both endothelial and smooth muscle cells (p = 0.05). This is the first study using skin biopsy slices to examine exposure of cetacean tissue to a CYP1A1 inducer. It demonstrates a causal relationship between chemical exposure and CYP1A1 induction and therefore validates the use of CYP1A1 expression in skin biopsies as a biomarker in cetaceans. Our protocol can be adapted to the investigation of chemicals, mixtures, concentrations, incubation times, or biological endpoints of choice. This should prove particularly relevant for these and other protected species that cannot be studied in the laboratory.